Aquaculture is now a major industry in the United states and the world. Many species grown in artificial environments require the addition of carotenoids to their diets, especially astaxanthin and canthaxanthin. It has been thought by those familiar in the industry that carotenoids natural to the species' environment are preferable to synthetic pigments. The production of natural carotenoids from natural sources such as phaffia yeast and marine microbial algae and crawfish shell address this industry need for natural pigments.
It is also of importance to find economically feasible means to dispose of various pigment bearing waste streams such as those of crawfish, crab, and now, sea cucumber gut material. Because the sea cucumber gut material is now known to be a rich source of usable pigments, there existed a potential to produce those isolated pigments and related lipid fractions from the sea cucumber industry waste materials provided that a method suitable for a large-scale operation could be found.
Carotenoids from sea cucumbers have been mentioned in various scientific journal articles, to wit:
Matsuno, et al reported the occurrence of astaxanthin as the major carotenoid in the gonads of the sea cucumbers Holothuria leucospilota and Stichopus japonicus, and beta carotene, echinenone, canthaxanthin and zeaxanthin were identified from the gonad of H. leucospilota and S. japonicus. On the other hand, astaxanthin and the esters, canthaxanthin, phoenicoxanthin, and ochincnone, were isolated by Bullock and Dawson from the red body wall of Psulus fabrichii. M. Tsushima, et al reported on the novel marine di-Z carotenoids, cucumariaxanthins A, B and C from the Sea cucumber Cucumbaria japonica. J. Findlay, et al reported on canthaxanthin from the species, Cucumaria frondosa. None of these reports provide any means whereby to utilize such carotenoids for any aquaculture or medical or healthfood industry, and none provide methods to concentrate such pigmented lipid fractions. PA0 a. Separating gut material from the processed sea cucumber by hand or machine. Hand separating involves cutting the animal with a knife and physically removing the intestines. Machine cutting involves any mechanized embodiment wherein automated cutting tools are able to cut open the animal whereby the intestines are available for removal. PA0 b. The proteinaceous/lipid gut material is pretreated after separation from the animal with sufficient acid to lower the pH of the material to within a range of from about 4 to about 5.5 preferably about 4.3 to about 4.7 to suppress microbial degradation, demineralize the mass and improve overall red carotenoid pigment recovery during the subsequent extraction with solvent or combinations of solvents. PA0 c. In one method of processing, the proteinaceous/lipid gut material is mixed with acetone, alcohol or similar solvent in 3:1 solvent to material ratio and then agitated, for 24 hours or until sufficient pigment has released from the gut material to the solvent. PA0 d. The acetone or other solvent is decanted off of the remaining gut material and the gut material is washed subsequently four or more times with clean solvent to remove remaining carotenoids. PA0 e. The remaining gut material is centrifuged to purge remaining solvent, or heated in a closed vessel and the solvent reclaimed by methods known to those in the arts. PA0 f. The pigment bearing acetone or solvent is pumped to a `wiped film evaporator` as is known in the oil chemists' arts and the pigment is retained and sequestered and the solvent is reclaimed. PA0 g. The resultant lipid pigment fraction is a mixed carotenoid of canthaxanthin, cucumariaxanthin, astaxanthin, and zeaxanthin. The major carotenoid is canthaxanthin in the example of Cucumaria frondosa. PA0 h. The resultant pigmented lipid fraction can be stabilized with known antioxidants as are known in the arts. PA0 g. The remaining gut material is then dried and extracted with hexane, butane, supercritical carbon dioxide, or similar solvent to remove a golden oil essentially free of pigments. The resultant remaining dry proteinaceous material free of lipids is high in protein and polysaccharides and is suitable for inclusion into animal feeds. This proteinaceous de-lipidized meal is unique in the marine fish-meal industry inasmuch as it is both de-pigmented, de-lipidized, is high in usable protein, and is essentially free of odoriferous contaminants. It is easily manufactured into powder. PA0 a. Subjecting the dried gut material which has a moisture content of less than approximately 10% to a standard "oil-recovery procedure" with hexane or other suitable solvent known to those skilled in the arts, wherein the dry gut material is mixed at 1:1 ratio at 130 F. for 5 minutes and then drained. The extracted product is then washed three times with fresh solvent. The solutions containing the carotenoid is desolventized in a wiped film (Luwa and Pope) evaporator at 95 C. and 28 inches Hg vacuum. PA0 b. Sequestering the solvent/lipid phase into containers designed for such use and freeing said solvent/carotenoid mixture from carotenoid oil phase by standard "steam stripping" or by "wiped film" oil technology. PA0 c. Treating such oil phase with anti-oxidants which are known to those skilled in the arts such as 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline (Ethoxyquin, Monsanto) at between 300 to 600 parts per million or vitamin E at approximately 1 percent. PA0 d. Subjecting the red pigmented oil thus obtained to a degumming process by the addition of 1% water to the oil then allowing to hydrate for 30 mintes and then centrifuging at 8000 rpm at 150 F. PA0 e. Recovering the concentrated pigment in the following manner: PA0 f. Determining the carotenoid content of extracted concentrate by standard HPLC methods known to those skilled in the arts. Concentrated pigment extracted by this present invention is suitable for inclusion in aquatic diets and contains parts per million of carotenoids canthaxanthin at between 2,000 and 5,000, depending on the carotenoid content of the original material. PA0 g. Stabilizing de-pigmented lipid from the carotenoid extraction procedure by the addition of vitamin E or other suitable antioxidants known to those in the arts, at between 1 and 5% by weight. PA0 h. Encapsulating de-pigmented lipid fraction from the carotenoid extraction procedure of sea cucumber intestinal mass subsequently in "soft-gel" capsules of 500 to 1000 milligrams, or packaging into suitable containers for oral and topical administration to mammals. Topical administration products can be obtained by any combination of emollients combined with the non-pigmented lipid fraction, depending on desired effect in a specific or general condition. PA0 g. Incorporating said pigmented lipid fraction, depending upon parts per million of carotenoid, into aquatic diets at a percentage of such food sufficient to produce carotenoid deposition into flesh or skin.
The U.S. Pat. No. 4,692,280 by Spinelli, Stout, and Nilsson describe a method for obtaining purified fish oils using supercritical carbon dioxide, and is included herein by reference. This patent does not disclose a means of obtaining sea cucumber lipid fraction purification through the supercritical purification methodologies set forth in the patent. U.S. Pat. No. 4,495,207 provides a method of extracting lipids from corn germ. Essentially the same methodologies were used by the inventor to extract lipids from sea cucumber tissue as is set forth in U.S. Pat. No. 4,495,207 and is included herein by reference.
Thus, there is no prior art for the efficient and large scale removal of lipid fractions, and non directed at obtaining usable carotenoid or non carotenoid lipid fractions of sea cucumber tissue.